CCNI Antibody

What is CCNI and why is it important in research?

CCNI (Cyclin I) is a member of the cyclin protein family with distinct structural and functional characteristics. In humans, the canonical CCNI protein consists of 377 amino acid residues with a molecular mass of approximately 42.6 kDa . Its primary subcellular localization is in the nucleus, with up to two different isoforms reported .

Unlike better-characterized cyclins that directly regulate cell cycle progression, CCNI has specialized functions that include involvement in spermatogenesis . It shows high expression in adult heart, brain, and skeletal muscle tissues, suggesting tissue-specific functions beyond basic cell cycle control .

CCNI is known by several synonyms in scientific literature, including CYC1, CYI, cyclin ITI, and CCNI1 . Evolutionary conservation is evident across numerous species, with CCNI gene orthologs identified in mouse, rat, bovine, frog, zebrafish, chimpanzee, chicken, camel, and woodpecker models .

This protein's unique expression patterns and potential functions make it an important target for research into specialized cellular processes, particularly in tissues where it shows high expression.

What applications are most suitable for CCNI antibodies?

CCNI antibodies support multiple research applications, each with specific methodological considerations:

Western Blotting

Western blotting represents the most widely used application for CCNI antibodies, allowing detection of the 41-43 kDa protein in cell and tissue lysates . For optimal results:

• Use 10% SDS-PAGE gels for proper separation of CCNI (predicted band size: 41-43 kDa)

• Load approximately 30 μg of whole cell lysate per lane

• Apply primary antibody at recommended dilutions (typically 1:1000)

• Include positive controls such as 293T whole cell lysates, which consistently show CCNI expression

Immunohistochemistry (IHC)

For tissue localization studies:

• Use recommended dilutions of 1:50-1:200

• Select appropriate fixation and antigen retrieval methods

• Include positive control tissues (heart, brain, skeletal muscle)

• Consider tissue-specific expression patterns when interpreting results

Immunofluorescence (ICC-IF)

For subcellular localization studies:

• Apply antibodies at 0.25-2 μg/mL concentration

• Include appropriate nuclear counterstains to confirm CCNI's nuclear localization

• Consider confocal microscopy for precise localization analysis

ELISA

For quantitative detection of CCNI:

• Both direct and sandwich ELISA formats are applicable

• Standardize with recombinant CCNI protein when available

• Optimize blocking conditions to minimize background

Each application requires specific validation steps and controls to ensure reliable results when working with CCNI antibodies.

How should researchers validate CCNI antibodies for experimental use?

Thorough validation is essential for ensuring reliable results with CCNI antibodies. A comprehensive validation strategy should include:

Specificity Validation

• Positive and negative controls:

  • Use tissues with known high CCNI expression (heart, brain, skeletal muscle)

  • Include negative controls (tissues with minimal CCNI expression)

  • Consider CCNI knockdown samples as specificity controls

• Western blot validation:

  • Confirm the observed molecular weight matches the predicted 41-43 kDa size

  • Test antibodies targeting different CCNI epitopes

  • Perform peptide competition assays when the immunizing peptide is available

• Cross-reactivity assessment:

  • Test reactivity against related proteins

  • Evaluate potential cross-species reactivity when working with non-human models

Performance Validation

Functional Validation

• Overexpression studies: Confirm increased signal with CCNI overexpression

• Knockdown studies: Verify signal reduction following CCNI-targeted siRNA treatment

• Cross-technique concordance: Compare results across different detection methods

Documenting validation results thoroughly ensures experimental reliability and facilitates troubleshooting when unexpected results occur.

What epitope considerations affect CCNI antibody selection?

The choice of epitope significantly impacts CCNI antibody performance in different applications and experimental contexts:

Epitope Positioning

CCNI antibodies target different regions of the protein:

• N-terminal region antibodies (e.g., Aviva Systems Biology's ARP64388_P050)

• C-terminal region antibodies (e.g., those targeting sequences like "VYRPLKHTLVTCDKGVFRLHPSSVPGPDFSKDNSKPEVPVRGTAAFYHHLPAASGCKQTSTKRKVEEMEVDDFYDGIKRLYNEDNV")

These different epitope targets affect:

• Accessibility in native versus denatured conditions

• Potential cross-reactivity with related proteins

• Sensitivity to post-translational modifications

Functional Domain Awareness

When selecting antibodies, consider CCNI's functional domains:

• Cyclin box region (critical for potential CDK interactions)

• Regions involved in protein-protein interactions

• Sites of known post-translational modifications

Cross-Species Applications

For research involving multiple species:

• Epitopes in highly conserved regions provide broader cross-reactivity

• Compare the immunogen sequence to the target species sequence

• Species-specific reactivity varies (e.g., some antibodies are validated for human, mouse, rabbit, rat, bovine, dog, guinea pig, and hamster samples)

Practical Selection Guidelines

• For structural studies: Choose antibodies targeting accessible epitopes

• For functional studies: Select antibodies that don't interfere with important functional domains

• For detecting specific isoforms: Choose antibodies targeting isoform-specific regions

• For detecting post-translational modifications: Avoid antibodies targeting modification sites

Understanding these epitope considerations helps researchers select the most appropriate CCNI antibody for their specific experimental requirements.

How can different types of CCNI antibodies (monoclonal vs. polyclonal) affect experimental outcomes?

The choice between monoclonal and polyclonal CCNI antibodies significantly impacts experimental results based on their distinct characteristics:

Polyclonal CCNI Antibodies

Polyclonal antibodies, such as the rabbit polyclonal anti-CCNI antibodies offered by Atlas Antibodies , provide:

• Recognition of multiple epitopes on CCNI protein

• Generally higher sensitivity due to multiple binding sites

• Greater tolerance to minor protein denaturation or modifications

• Potential batch-to-batch variability

• Possible higher background in some applications

Monoclonal CCNI Antibodies

Monoclonal antibodies offer:

• Recognition of a single specific epitope

• Consistent reproducibility between batches

• Often higher specificity for particular applications

• Potentially lower sensitivity for low-abundance targets

• More vulnerability to epitope masking or modification

Application-Specific Considerations

Experimental Strategy Using Both Types

For critical experiments, consider using both antibody types in complementary approaches:

• Use polyclonal antibodies for initial detection and localization

• Confirm findings with monoclonal antibodies targeting specific epitopes

• Validate results across multiple methodologies

This strategic approach leverages the strengths of each antibody type while compensating for their respective limitations.

What are the best practices for optimizing Western blot procedures with CCNI antibodies?

Successful Western blot detection of CCNI requires optimization of several critical parameters:

Sample Preparation

• Extraction buffer: RIPA buffer with protease inhibitors effectively extracts nuclear proteins like CCNI

• Protein concentration: 20-30 μg total protein per lane is typically sufficient

• Sample handling: Minimize freeze-thaw cycles to prevent degradation

• Denaturation: Heat samples at 95°C for 5 minutes in reducing sample buffer

Gel Electrophoresis Parameters

• Gel percentage: 10% SDS-PAGE gels provide optimal resolution around CCNI's molecular weight (41-43 kDa)

• Running conditions: 100-120V constant voltage for uniform migration

• Include appropriate molecular weight markers spanning 25-75 kDa range

Transfer Conditions

• Transfer method: Wet transfer at 100V for 1 hour or 30V overnight at 4°C

• Membrane selection: PVDF membranes generally provide better results than nitrocellulose for CCNI

• Transfer validation: Use reversible staining (Ponceau S) to confirm successful transfer

Antibody Incubation

• Blocking: 5% non-fat milk in TBST (1 hour at room temperature)

• Primary antibody dilution: Typically 1:1000 in 5% BSA/TBST for most CCNI antibodies

• Primary antibody incubation: Overnight at 4°C with gentle rocking

• Washing: 3-5 washes with TBST, 5-10 minutes each

• Secondary antibody: Anti-rabbit HRP conjugate at appropriate dilution (typically 1:5000-1:10000)

Signal Detection

• Detection method: Enhanced chemiluminescence (ECL) with appropriate exposure times

• Expected band pattern: Primary band at approximately 41-43 kDa

• Quantification: Normalize CCNI signals to appropriate loading controls (β-actin, GAPDH)

Troubleshooting Common Issues

Following these optimized protocols helps ensure consistent and reliable detection of CCNI by Western blotting.

How do CCNI antibodies perform across different species?

CCNI is well-conserved across species, affecting antibody cross-reactivity in comparative studies:

Cross-Species Reactivity Profiles

Commercial CCNI antibodies show varying species reactivity profiles:

• Human-specific antibodies: Optimized for human samples only

• Multi-species antibodies: Some react with human, mouse, rabbit, rat, bovine, dog, guinea pig, and hamster samples

• Specialized antibodies: Some are developed for specific research models like camel or woodpecker

Sequence Conservation Analysis

CCNI sequence conservation affects antibody performance:

• Mammalian species show high sequence homology (typically >90% with human CCNI)

• Non-mammalian vertebrates show more divergence

• Conservation is highest in functional domains like the cyclin box

Experimental Validation Approaches

When working with new species:

• Perform comparative Western blots with known positive controls

• Include relevant negative controls

• Test multiple antibodies targeting different epitopes

• Optimize protocols for each species (antibody concentration, incubation time)

Species-Specific Considerations

Understanding these cross-species considerations is essential for comparative studies and when translating findings between model systems and human applications.

What are the methodological approaches for studying CCNI subcellular localization?

Investigating CCNI's subcellular localization, primarily in the nucleus , requires specialized methodological approaches:

Immunofluorescence Methodology

For precise subcellular localization:

• Cell preparation:

  • Fixation: 4% paraformaldehyde (10 minutes at room temperature)

  • Permeabilization: 0.1-0.5% Triton X-100 (5-10 minutes)

  • Blocking: 1-5% BSA in PBS (30-60 minutes)

• Antibody application:

  • Primary CCNI antibody: 0.25-2 μg/mL concentration

  • Secondary antibody: Fluorophore-conjugated at manufacturer's recommended dilution

  • Nuclear counterstain: DAPI or Hoechst dyes

• Imaging parameters:

  • Confocal microscopy for precise colocalization

  • Z-stack acquisition for 3D localization analysis

  • Multi-channel acquisition for colocalization studies

Biochemical Fractionation Approaches

For quantitative assessment of CCNI distribution:

• Subcellular fractionation protocol:

  • Separate nuclear and cytoplasmic fractions

  • Confirm fraction purity with markers (Lamin B for nuclear, GAPDH for cytoplasmic)

  • Analyze fractions by Western blotting with CCNI antibodies

• Quantitative analysis:

  • Densitometric quantification of CCNI in each fraction

  • Normalization to fraction-specific loading controls

  • Calculation of nuclear/cytoplasmic distribution ratios

Colocalization Studies

For understanding CCNI relationship with nuclear structures:

• Dual immunofluorescence with markers for:

  • Nuclear speckles (SC35)

  • Nucleoli (Fibrillarin)

  • Chromatin states (Histone modifications)

  • Nuclear matrix (Lamin proteins)

• Colocalization analysis:

  • Calculate Pearson's or Mander's coefficients

  • Perform line scan analysis across nuclear structures

  • Implement 3D rendering for spatial relationship analysis

These methodological approaches provide complementary information about CCNI localization, offering insights into its potential nuclear functions.

How can researchers investigate CCNI's role in spermatogenesis?

CCNI is known to be involved in spermatogenesis , and antibody-based approaches enable detailed investigation of this function:

Developmental Expression Analysis

Track CCNI expression throughout spermatogenesis:

• Immunohistochemistry approach:

  • Process testicular sections with CCNI antibodies (1:50-1:200 dilution)

  • Implement stage-specific analysis of seminiferous tubules

  • Quantify expression across developmental stages

• Western blot analysis:

  • Compare CCNI levels across developmental timepoints

  • Fractionate testicular cells to analyze specific populations

  • Correlate with known spermatogenesis markers

Cell Type-Specific Localization

Identify which spermatogenic cells express CCNI:

• Dual immunofluorescence with cell-specific markers:

  • Spermatogonia: PLZF, OCT4

  • Spermatocytes: SYCP3, γH2AX

  • Spermatids: PRM1, TNP1

• High-resolution imaging:

  • Confocal microscopy for precise colocalization

  • Super-resolution techniques for detailed subcellular distribution

Functional Studies

Assess CCNI's functional significance in spermatogenesis:

• In vitro approaches:

  • CCNI knockdown in spermatogenic cell cultures

  • Assessment of meiotic progression

  • Evaluation of chromatin dynamics and DNA damage repair

• Protein interaction networks:

  • Co-immunoprecipitation with CCNI antibodies in testicular lysates

  • Identification of testis-specific binding partners

  • Validation of interactions with proximity ligation assays

Comparative Analysis

Understand evolutionary conservation of CCNI function:

• Cross-species comparison:

  • Analyze CCNI expression and function across rodent and human samples

  • Correlate with known fertility phenotypes

  • Integrate with genomic data from infertility studies

These methodological approaches provide comprehensive insights into CCNI's specific role in the complex process of spermatogenesis.

What methods can be used to investigate CCNI protein-protein interactions?

Understanding CCNI's functional networks requires comprehensive investigation of its protein-protein interactions:

Co-Immunoprecipitation (Co-IP) Strategies

• Standard Co-IP protocol:

  • Lyse cells in non-denaturing buffer to preserve protein complexes

  • Immunoprecipitate CCNI using 2-5 μg antibody per mg protein lysate

  • Analyze co-precipitated proteins by Western blot or mass spectrometry

  • Validate with reciprocal IP using antibodies against identified partners

• Crosslinking-assisted Co-IP:

  • Implement mild crosslinking (0.5-1% formaldehyde, 10 minutes)

  • Stabilize transient interactions before lysis

  • Perform IP with CCNI antibodies and analyze complexes

  • Reverse crosslinks before final analysis

Proximity-Based Interaction Methods

• Proximity Ligation Assay (PLA):

  • Incubate fixed cells with CCNI antibody and partner protein antibody

  • Apply species-specific PLA probes with oligonucleotides

  • Perform rolling circle amplification where probes are in proximity (<40 nm)

  • Visualize discrete interaction sites as fluorescent spots

• Advanced microscopy approaches:

  • FRET analysis with labeled antibody fragments

  • Live-cell imaging with proximity sensors

  • Super-resolution microscopy for detailed interaction mapping

Domain-Specific Interaction Analysis

Identify critical binding regions:

• Use antibodies targeting specific CCNI domains

• Compare immunoprecipitation profiles with various antibodies

• Map interaction interfaces through epitope competition experiments

Stimulus-Dependent Interaction Profiling

Understand dynamic interaction networks:

• Map CCNI interactome changes following:

  • Cell cycle progression

  • Differentiation signals

  • Stress conditions

• Quantify interaction dynamics through sequential co-IPs

These methods provide complementary information about CCNI's protein interaction network, offering insights into its functional role in cellular processes.

How can CCNI antibodies be used to study post-translational modifications?

Post-translational modifications (PTMs) often regulate protein function, and antibody-based approaches enable their investigation in CCNI:

Phosphorylation Analysis

• Phospho-detection strategies:

  • Immunoprecipitate CCNI and probe with phospho-specific antibodies

  • Perform lambda phosphatase treatment as control

  • Use Phos-tag™ gels to detect mobility shifts in phosphorylated CCNI

• Site-specific phosphorylation:

  • Develop antibodies against predicted phosphorylation sites

  • Validate with phosphomimetic and phospho-null mutants

  • Map phosphorylation dynamics across cellular conditions

Ubiquitination Analysis

• Modified CCNI detection:

  • Immunoprecipitate CCNI under denaturing conditions

  • Probe with ubiquitin-specific antibodies

  • Detect characteristic ladder pattern of ubiquitinated proteins

• Stability assessment:

  • Treat cells with proteasome inhibitors

  • Monitor CCNI levels by Western blotting

  • Correlate ubiquitination status with protein stability

Other Modification Types

• Acetylation assessment:

  • Immunoprecipitate CCNI and probe with acetyl-lysine antibodies

  • Treat cells with HDAC inhibitors to enhance acetylation signals

  • Map acetylation sites through mass spectrometry after immunopurification

• SUMOylation analysis:

  • Immunoprecipitate CCNI under denaturing conditions

  • Probe with SUMO-specific antibodies

  • Identify SUMO-modified forms through mobility shift detection

Functional Correlation

Link modifications to functional outcomes:

• Correlate PTM status with CCNI localization

• Assess protein-protein interaction dependencies on PTMs

• Evaluate protein stability and turnover relative to modification status

Technical Considerations

These approaches provide comprehensive analysis of CCNI post-translational modifications and their functional significance.

What approaches can be used to study CCNI in the immune system?

While the search results don't specifically mention CCNI's role in the immune system, researchers can adapt antibody-based approaches to investigate potential immune functions:

Expression Analysis in Immune Tissues and Cells

• Tissue expression profiling:

  • Use CCNI antibodies to screen lymphoid tissues (thymus, spleen, lymph nodes)

  • Implement immunohistochemistry at 1:50-1:200 dilutions

  • Quantify expression levels across different immune compartments

• Immune cell-type profiling:

  • Perform flow cytometry with CCNI antibodies on immune cell populations

  • Analyze expression in T cells, B cells, macrophages, dendritic cells

  • Sort cells based on CCNI expression for functional characterization

Functional Analysis in Immune Responses

• Activation-dependent expression:

  • Stimulate immune cells with antigens, cytokines, or pathogens

  • Monitor CCNI expression changes by Western blot

  • Correlate with activation markers and functional outcomes

• Knockdown/knockout approaches:

  • Deplete CCNI in immune cells using siRNA or CRISPR

  • Assess effects on proliferation, cytokine production, differentiation

  • Perform immune function assays (e.g., T cell proliferation, antibody production)

Integration with Immune Signaling

• Co-immunoprecipitation studies:

  • Identify CCNI interactions with immune signaling components

  • Map dynamic changes in protein complexes during immune activation

  • Validate functional significance through targeted disruption

• Phosphorylation analysis:

  • Examine CCNI phosphorylation status during immune signaling

  • Identify responsible kinases in immune pathways

  • Correlate modifications with functional outcomes

This methodological framework provides a comprehensive approach to investigating CCNI's potential roles in immune system function, which remains an open area for research investigation.